Proper controls are essential to help ensure success in every RNAi experiment. The number and types of controls chosen depend upon the ultimate research goal (see table below). We have simplified control reactions by providing a selection of RNAi technologies designed to assist researchers in identifying and validating drug targets, generating publishable data, and submitting grants. Our RNAi controls allow you to:

See below for guidance on multiple siRNA sequences to the same target, titration of RNAi, untransfected controls, and downstream controls.

Transfection controls

With overexpression experiments, even low transfection efficiencies can often yield results. In contrast, for gene knockdown to be measurable in a cell population it is important to have the highest transfection efficiency possible. Even small reductions in transfection efficiency can limit your ability to identify functional differences in your experimental samples or validate knockdown by qRT-PCR or western blot analysis.

To achieve the highest transfection efficiency possible, particularly for gene knockdown experiments, first optimize transfection conditions for your cell lines. Keep in mind that it is also important to monitor variation in transfection from experiment to experiment.

There are several transfection reagents to choose from, depending on cell type and other factors. You may monitor transfection efficiency using these reagents paired with fluorescently labeled siRNA duplexes.

Negative controls

Negative control siRNAs—siRNAs with sequences that do not target any gene product—are essential for determining the effects of siRNA delivery and for providing a baseline to compare to siRNA-treated samples. Negative controls are available for Silencer® Select siRNA, Stealth RNAi™ siRNA, and Silencer® siRNA. Learn more about the differences between these types of siRNA.

We recommend using one or more negative controls in every RNAi experiment. You should also match the control siRNA to the experimental siRNA type (for example, use only Silencer® Select siRNA controls for experiments using Silencer® Select siRNA).

The two Silencer® Select Negative Control siRNAs include the same modifications for reducing off-target effects as found in other Silencer® Select siRNAs and have no significant sequence similarity to mouse, rat, or human gene sequences. These negative control siRNAs have been tested by microarray analysis and shown to have minimal effects on gene expression. In addition, these controls have been tested in multiparametric cell-based assays and are proven to have no significant effect on cell proliferation, viability, or morphology in the cell lines tested.

Stealth RNAi™ siRNA negative controls

For experiments using Stealth RNAi™ siRNA, we have predesigned negative controls with the following features:

Three levels of GC content to match that of the experimental Stealth RNAi™ siRNAs: high, medium, and low

No homology to any known vertebrate gene

Tested sequences for several duplexes, which do not induce a stress response

In addition, by using the BLOCK-iT™ RNAi Designer you can also choose scrambled controls for any Stealth RNAi™ siRNA sequence. We also provide a negative control in our RNAi vector cloning kits. The negative control should be of the same chemical structure as the target RNAi molecules. For example, if you are using shRNA vectors, the negative control should have the same vector backbone but a different RNAi sequence. Learn more about using Stealth RNAi™ siRNA controls.

Silencer® siRNA negative controls

Ambion® Silencer® Negative Control siRNAs have no significant sequence similarity to mouse, rat, or human gene sequences. Theses controls have also been tested in cell-based screens and proven to have no significant effect on cell proliferation, viability, or morphology.

Nontargeting with limited sequence similarity to known genes

Validated for use in human, mouse, and rat cells

Functionally proven to have minimal effects on cell proliferation and viability

HPLC purified, duplexed, and ready to use

Positive controls

Positive controls provide more confidence in your RNAi experiments by helping to confirm that experimental conditions were met to achieve robust data. Positive controls are siRNA molecules that are known to achieve high levels of knockdown (>70%). A positive control should be used to optimize siRNA delivery conditions and to reconfirm high levels of delivery in each RNAi experiment. When a positive control fails to produce the anticipated phenotype, carefully evaluate your experimental conditions and decide if some factors need to be adjusted. Examples of positive controls are genes expressed at easily detectable levels, such as p53, lamin, and GAPDH. Positive controls are available for Silencer® Select siRNA, Stealth RNAi™ siRNA, and Silencer® siRNA. Learn more about the differences between these types of siRNA.

You should also match the control siRNA to the experimental siRNA type (for example, use only Silencer® Select siRNA controls for experiments using Silencer® Select siRNA).

Rescue experiments

RNAi rescue experiments are performed to help ensure that the observed effect is due to knockdown of the target gene of interest. If you are using an inducible RNAi vector system, turn off the RNAi expression by removing tetracycline from the medium. If you are using Silencer® Select siRNA or Stealth RNAi™ siRNA, there are two main methods used to rescue the phenotype. The first method involves designing RNAi sequences to the 3’ UTR and then transfecting the cells after knockdown with a vector expressing the open reading frame (ORF) of the gene of interest. In the second method, if the RNAi sequences were designed to the ORF, you can use GeneArt® gene synthesis or a mutagenesis kit to create one or more silent third-codon point mutations within the region targeted by the RNAi sequence, preferably the seed and cleavage regions on the antisense strand (bases 2–12).

Additional RNAi control guidelines

Multiple siRNA sequences to the same target

siRNA sequences with partial homology to other targets may contribute to off-target activity. Gene profiling experiments have shown that duplexes with partial homology to other transcripts can cleave the target or act like a microRNA (miRNA), inhibiting translation of the target mRNA. Specificity studies have revealed that siRNA duplexes can have varying activities depending on the number, position, and base pair composition of mismatches with respect to the target RNA. To ensure that knockdown of the intended gene causes a particular siRNA phenotype, the phenotypic results should be confirmed by at least two siRNA molecules that target non-overlapping regions of the target mRNA. Thus, if one siRNA sequence produces a particular phenotype but the second siRNA sequence (designed to target the same gene) produces a different phenotype, then you cannot conclude that the gene of interest was successfully knocked down.

Titration of siRNA

Silencer® Select siRNA and Stealth RNAi™ siRNA can be very effective even at low concentrations, and you should aim to use the lowest effective level to avoid altering the cells’ normal processes. Titrating down the dose of the Silencer® Select siRNA and Stealth RNAi™ siRNA duplex enables you to reduce any off-target or nonspecific effects while achieving robust knockdown.

Untransfected controls

Silencer® Select siRNA and Stealth RNAi™ siRNA can be very effective even at low concentrations, and you should aim to use the lowest effective level to avoid altering the cells’ normal processes. Titrating down the dose of the Silencer® Select siRNA and Stealth RNAi™ siRNA duplex enables you to reduce any off-target or nonspecific effects while achieving robust knockdown.

Downstream controls

Before transfecting cells and performing qRT-PCR and western blots to measure mRNA and protein levels, we recommend validating your downstream reagents. Validating qRT-PCR primers or antibodies for your positive control and target genes before performing knockdown experiments in your cell line helps to ensure that your reagents are sensitive enough to detect changes in your target gene’s expression due to knockdown. Without sufficient sensitivity, it can be difficult to interpret knockdown results from genes or proteins with low expression levels.